We propose to extend our studies of Triton Shells of intact erythrocytes to answer a number of important questions about the organization of the erythrocyte cytoskeleton and its interaction with membrane glycoproteins. Triton shells are prepared by mixing intact erythrocytes with Triton suspensions in isotonic media and sedimenting the unextracted cytoskeleton into a sucrose gradient to separate it from cytoplasm, detergent, solubilized lipids and proteins. This procedure provides the best available method for preparing cytoskeletal components without hypotonic extraction which may denature them. First, the protein composition of the shells as a function of Triton concentration will be analyzed in detail by two-dimensional gel electrophoresis and correlated with the lipid content of the shells. Second, the shells will be disassembled using ionic and enzymatic treatments in order to determine the bases for their interactions. The understanding of the component interactions will then be tested in reconstitution studies. Third, integral membrane protein linkages to the cytoskeleton will be measured as a function of ionic composition changes, antibody crosslinking of glycoproteins and depletion of cytoplasmic ATP. Fourth, the technology and knowledge gained in the above studies will be applied to understand the effect of in vivo and in vitro aging of erythrocytes and Vitamin E therapy on the organization of the cytoskeleton and the interaction of integral membrane proteins with it. Finally, red cell membrane mutants, hereditary spherocytosis and hereditary elliptocytosis, will be studied to understand the molecular bases of the defects. From this study we will obtain knowledge about membrane structure, the mechanisms of cytoskeletal-membrane interaction, aging effects on membrane structure, and the organization of the erythrocyte cytoskeleton in normal and disease states.